The fluorescein structural motif and one step cyclization from phthalic anhydride and resorcinol is believed to have been first described in 1871 by Baeyer (Berichte. 1871: 4, 555). Graebe (Annalen. 1887: 18, 318) is believed to be the first to use halogenated phthalic anhydride as substrates in the cyclization noting in his report the use of an excess of anhydride (1.3 equivalents) to resorcinol. Iodination of dichlorofluorescein appeared in the literature in 1887 with a report by Le Royer (Annalen. 1887: 238, 359). In the 20th century, several uses for fluorescein analogs emerged. The compounds have been used as textile dyes, biological stains, building blocks for non-volatile memory devices, thermoimaging substrates and food and cosmetics coloring. For example, erythrosine (FD&C No. 3) and partially iodinated erythrosine (D&C Nos. 11 and 12) are used as food, drug and cosmetic dyes. A particular tetraiodo-xanthene, Rose Bengal, has been used for visualization of ocular disease and, in radiolabeled form, as a medical diagnostic for liver function, appearing in the United States Pharmacopeia in 1965.
The cyclization, however, to create the xanthene core of Rose Bengal has not substantially improved from the 1880's technology (high temperature melts in open kettles), even though interest in the synthesis of the non-halogenated analogs and elaboration on the fluorescein motif is extensive. The known synthetic methods produce a range of unpredictable and poorly characterized impurities including residual solvents, inorganic compounds and organic compounds derived from side reactions or degradation processes. For many historical uses in industrial applications, food dyes or diagnostics, these impurities are permissible. For example, the United States Code of Federal Regulations (CFR) allows an impurity level for FD&C No. 3 (erythrosine) of no more than 1% mono-iodinated impurities and no more than 9% of other lower iodinated fluoresceins. The CFR also allows residual impurities originating in the cyclization step, such as partially iodinated phthalic acids and resorcinols (for example, see: Kamikura, Shokuhin Eiseigaku Zasshi 1985: 26, 243 and Wada et al., Food Add. Contam. 2004: 21, 1137).
Such historical coloring agent specifications are quite disparate to modern International Conference of Harmonisation (ICH) guidelines for a new drug substance, which requires reporting of impurities of 0.05% or higher, comprehensive identification of any organic impurity present at levels of 0.1% or higher, and thorough toxicologic qualification of any impurities over 0.15%, and further provide limits on inorganic impurities and especially stringent limits on residual solvents. Hence, when introducing this class of compounds into the body at therapeutic doses, the necessity to have a well controlled, predictable and reproducible synthesis becomes a priority. Unpredictable generation of multiple impurities during synthetic steps or in purification is not an option for inclusion in such a specification, especially with a potential parenteral drug product formulation.
To make reagent grade Rose Bengal, the United States Pharmacopeia XXII recommends using HCl to purify Rose Bengal via an acid/base manipulation. The present inventors have found that, quite surprisingly, when oxidants like hydrogen peroxide or oxone are present, treatment of iodinated fluoresceins with reagents that contain or can generate aqueous chloride ions causes a side reaction where one or more of the I substituents can be transhalogenated to Cl. This can also occur when chloride free radicals, hypochlorite ion, or hypochlorous acid are present. This side reaction during the preparation of Rose Bengal has not been reported previously and the cyclization step, the iodination step and any purification scheme must be carefully controlled to prevent this undesirable side reaction.
While iodinated fluorescein analogs have been previously described and often generically recite the word “halogen”, none of these prior disclosures appear to enable the synthesis of iodo-xanthene substituted fluoresceins directly from iodo-resorcinols. Also, none of the prior disclosures appear to require at least one iodine to be present in the molecule, and none claim a pharmaceutical applicability of these compounds that is most certainly iodine dependent. Predominant use of fluoresceins for nontherapeutic purposes have resulted in a paucity of information regarding the description and reduction to practice of methods required for the preparation of high purity active pharmaceutical ingredients in this compound class, as well as methods for the identification, characterization and synthesis of minor by-products which may have utility as human therapeutics or as colorants. Iodinated xanthenes have been generically included as embodiments in various disclosures, and Rose Bengal is a well known compound first described in the 1880's. None of these references, however, has described the isolation or identification, nor disclosed or indicated the possibility of the existence of, the transhalogenated minor products composed of at least one I and at least one Cl substituent on the xanthene core. The present inventors have discovered that these products can exist in up to 2% by weight in commercial samples of Rose Bengal. Furthermore, prior references allude to lower iodinated xanthene contaminants (for example, not more than 9% is allowed for the dye certification of erythrosine in the present CFR 74.303), but none of these references have proposed a structure or a name for triiodinated versions that can be substituted with hydrogen in either the 2′ or 4′ position or their corresponding atropisomers (see FIGS. 1q and 1r infra and refer to U.S. Pat. No. 6,649,769 for a discussion of atropisomerism on this scaffold). Nor has the prior references taught or suggested the isolation or enabled the synthesis of independent I/Cl substitutions about the xanthene core. The present invention identifies, characterizes and establishes methods to efficiently control the synthesis of these by-products to meet the standards required for utility in pharmaceutical applications. In addition, the process of the present invention avoids undesirable formation of these by-products, avoids the necessity of using multiple solvents and sets strict control of reagents, all of which improve handling, yield, purity and applicability of the process for pharmaceutical use.